Induction of tumor-specific immunity enables long-term prevention of recurrence of tumors. Such immunotherapy, however, basically depends on the presence or absence of tumor specific antigens and on whether or not a cytotoxic immune response can be induced, in which an antigen is presented and tumor cells are recognized. Cytotoxic T lymphocytes (CTLs), together with costimulatory molecules, recognize MHC class I molecules complexed with peptides derived from cytoplasmic proteins presented on the cell surface (refer to e.g., non-patent literature 1). Tumor-specific antigens have been detected in various human tumors (refer to e.g., non-patent literatures 2and 3). Cancer vaccine therapy has focused either on the use of inactivated tumor cells or their lysates administered together with an adjuvant or a cytokine. It was recently reported that gene transfer of various cytokines, MHC molecules, costimulatory molecules, or tumor antigens to tumor cells enhances the visibility of tumor cells to immune effector cells (refer to e.g., non-patent literature 4). If induction of antitumor immunity becomes possible by direct intratumoral inoculation (in situ cancer vaccine) of HSV etc, major problems in using cancer vaccines for therapeutic purposes will be overcome. That is, the harvest of patients' autologous tumor cells, their in vitro manipulation such as culture and irradiation and identification of specific tumor antigens will not be needed for preparation of cancer vaccines.
HSV is a double-stranded DNA virus, contains the largest genome (153 kb) among DNA viruses that proliferate in nuclei, which encodes 84 kinds of open reading frames. The genome consists of the L (long) and S (short) components, each having a unique sequence flanked by inverted repeat sequences on its both sides. The complete nucleotide sequence of the viral genome has been determined and the functions of almost all viral genes have been elucidated. Martuza et al. developed herpes simplex virus mutant G207, a multi-gene mutant of herpes simplex virus type 1 (HSV-1) with a deletion in the γ34.5 gene and a lacZ gene insertion in the ICP6 gene (refer to e.g., non-patent literature 5). G207 is superior to other virus vectors from the therapeutic viewpoint. G207 is replicated in dividing cells, thereby causing lysis and death of infected cells, whereas in non-dividing cells, the virus proliferation is markedly weak. Inoculation with G207 into tumors established in athymia mice suppressed the tumor growth due to tumor-specific replication and prolonged the survival period of tumor-bearing mice (refer to e.g., non-patent literature 6). Further, in immune responsive mice, intratumoral inoculation with G207 induces tumor-specific immune responses, thereby suppressing the growth of tumors that have not been inoculated with G207 as well (refer to e.g., non-patent literature 7). In this case, G207 is acting as an in situ cancer vaccine (refer to e.g., patent literature 1). To date, gene therapy using herpes simplex virus mutant G207 has been performed, focusing on brain tumors, and their clinical application has also started in the U.S. (refer to e.g., non-patent literature 8).
The murine colon carcinoma cell line CT26 is poorly immunogenic and does not induce tumor-specific CTLs at the detectable level. CT26 is widely used as a syngeneic tumor model to study immunotherapy (refer to e.g., non-patent literatures 9 and 10). As a tumor-specific antigen in CT26, the MHC class I-restricted AH1 peptide, derived from an envelop protein (gp70) of an endogenous murine leukemia virus, was identified (refer to e.g., non-patent literature 10). It was confirmed that CT26 tumors which have been subcutaneously established can be treated by adoptive immunity cell transfer of peptide-specific CTLs and that there is a correlation between induction of tumor-specific CTLs and antitumor effects (refer to e.g., non-patent literature 10). To investigate the efficacy of HSV-1 mutant G207 as an in situ cancer vaccine, the inventors have used the poorly-immunogenic murine colon carcinoma cell line CT26, which expresses the tumor antigen identified. Further, the inventors have evaluated the efficacy of G207 using the syngeneic M3 mouse melanoma model to clarify that antitumor responses induced by intratumoral inoculation with G207 can be commonly used (refer to e.g., non-patent literatures 11 and 12).
On the other hand, following methods for inactivating viruses are known: (a) physical inactivation methods such as heat treatment (refer to e.g., non-patent literature 13), ultraviolet irradiation (refer to e.g., patent literature 2), γ-irradiation (refer to e.g., patent literature 3), electron beam irradiation (refer to e.g., patent literatures 4 and 5), pressure treatment (refer to e.g., patent literature 6), and energizing treatment (refer to e.g., patent literature 7); (b) chemical inactivation methods such as sterilization using phenol, formalin, alcohol, etc., alkaline treatment (refer to e.g., patent literature 8), contact with singlet oxygen, which consists of normal oxygen molecules excited electronically and being at the high state in energy (refer to e.g., patent literature 9), and deoxyribonuclease treatment; and (c)the combination of these physical inactivation methods and chemical inactivation methods (refer to e.g., patent literatures 10 and 11).
In addition, the following are described regarding HSVgD as vaccines against herpes viruses: methods of producing recombinant HSVgD (refer to e.g., patent literature 12), recombinant HSVgD vaccine (refer to e.g., patent literature 13), recombinant DNA encoding HSV-2gD and the protein (refer to e.g., patent literature 14), vaccine formulations consisting of HSVgD and 3-deacylated monophosphoryl lipid A (refer to e.g., patent literature 15), methods of producing recombinant HSVgD using insect cells (refer to e.g., patent literature 16), the HSVgD molecule consisting of 300 amino acid sequences (refer to e.g., patent literature 17), a vaccine composition containing HSVgD or an HBV antigen in conjunction with an adjuvant (refer to e.g., patent literature 18), and a fusion protein of HSVgB polypeptide and HSVgD (refer to e.g., patent literature 19). However, nothing has been known of using HSVgD for a cancer vaccine.    Patent literature 1: National Publication of International Patent Application No. 2001-513508    Patent literature 2: Japanese Patent Publication No. 45-9556    Patent literature 3: Japanese Laid-Open Application No. 2-9367    Patent literature 4: Japanese Laid-Open Application No. 4-200353    Patent literature 5: Japanese Laid-Open Application No. 4-92671    Patent literature 6: Japanese Laid-Open Application No. 6-142197    Patent literature 7: Japanese Laid-Open Application No. 2000-175682    Patent literature 8: Japanese Laid-Open Application No. 9-187273    Patent literature 9: Japanese Laid-Open Application No. 11-199490    Patent literature 10: Japanese Laid-Open Application No. 6-321994    Patent literature 11: National Publication of International Patent Application No. 8-504407    Patent literature 12: EP101655    Patent literature 13: EP-A-628633    Patent literature 14: WO90/13652    Patent literature 15: U.S. Pat. No. 6,027,730    Patent literature 16: EP-A-531728    Patent literature 17: U.S. Pat. No. 5,654,174    Patent literature 18: National Publication of International Patent Application No. 2002-506045    Patent literature 19: Japanese Patent No. 2999966    Non-patent literature 1: Annu. Rev. Immunol. 7, 445-480, 1989    Non-patent literature 2: Annu. Rev. Immunol. 12, 337-365, 1994    Non-patent literature 3: Adv. Immunol. 57, 281-351, 1994    Non-patent literature 4: Adv. Immunol. 58, 417-454, 1995    Non-patent literature 5: Nat. Med. 1, 938, 1995    Non-patent literature 6: Cancer. Res. 55, 4752, 1995    Non-patent literature 7: Hum. Gene Ther. 9, 2177-2185, 1999    Non-patent literature 8: Gene Ther, 7, 867-874, 2000    Non-patent literature 9: Cancer Res. 35, 2975, 1988    Non-patent literature 10: Proc. Natl. Acad. Sci. USA 93, 9730, 1996,    Non-patent literature 11: Hum. Gene Ther. 10, 385-393, 1999    Non-patent literature 12: J. Immunol. 160, 4457-4464, 1998    Non-patent literature 13: Thrombosis Research, 22, 233-238, 1981
Metastasis of cancer is a pathologic condition that is extremely difficult to treat; there have been no effective treatment to date. Although it has been reported that some limited number of chemotherapeutic drugs are efficacious, their side effects are regarded as questionable. In spite of recent drastic advancements in gene therapy using virus vectors, there are serious problems in terms of safety. An object of the present invention is to provide an antitumor agent, a tumor immunity inducer, a T cell activator, and a dendritic cell activator, which are extremely safe and capable of inducing an antitumor immune reaction enabling immunotherapy for a cancer in such a way that an antitumor effect on a human distant tumor such as a metastatic tumor is exhibited. Another object of the present invention is to provide, using such an agent, inducer, or activator, enhancement of an antitumor effect, a method for treating a distant tumor such as a metastatic tumor, a method for inducing tumor immunity, a method for identifying a tumor antigen, a method for activating T cells, and a method for activating dendritic cells.
The inventors have enthusiastically studied to solve the above-mentioned problems. Considering, in putting cancer therapy by virus vectors into practical use, safety in their use is a prerequisite, they prepared the inactivated HSV that was completely devoid of infectivity and has virus DNA destroyed, by subjecting the wild- type HSV to ultraviolet irradiation and heat treatment. They inoculated this inactivated HSV directly into malignant tumor tissues derived from the tumor cell line CT26 and found that a tumor-specific immune response was induced and malignant tumors have regressed. They also found that the inactivated HSV was able to similarly suppress the growth of distant malignant tumors that have not been directly inoculated and that the inactivated HSV activated human dendritic cells. Likewise, they inoculated HSVgD directly into malignant tumor tissues derived from the tumor cell line CT2 and found that a tumor specific immune response was induced and malignant tumors regressed and that the growth of distant malignant tumors that have not been directly inoculated was suppressed. They also found that HSVgD worked to activate T cells as a costimulatory factor for T cells and activated human dendritic cells. The present invention has been accomplished based on these findings.